BlaR1 is an integral-membrane b-lactam sensor protein involved with b-lactam resistance in pathogenic bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Specifically, binding of b-lactam (penicillin-like) antibiotics to the extracellular sensor domain of BlaR1 (BlaRS) initiates a transmembrane signal that stimulates the production of b-lactamase proteins, which then destroy the b-lactam antibiotics. The atomic-scale mechanism for the signal transduction remains poorly understood. The dominant hypothesis proposes an essential interaction between an extracellular loop (L2) and BlaRS, which breaks upon antibiotic binding and thus supplies the transmembrane signal. But the evidence of this interaction is conflicting and direct atomic-level evidence of this interaction is lacking.Accordingly, we used amide 15N spin relaxation and paramagnetic relaxation enhancement (PRE) to investigate the interaction between L2short – a peptide mimic of L2 – and BlaRS, and its response to b-lactam antibiotic binding by BlaRS. Our results indicate transient specific contacts between L2short and BlaRS at a site close to the active site pocket. This interaction involves residues in the b-sheet hairpin. Importantly, b-lactam binding does not disrupt the L2short-BlaRS interaction. Instead, our spin-relaxation studies suggest the b-sheet hairpin is intrinsically mobile, and that b-lactam binding leads to a redistribution of ps-ns backbone mobility of the b-sheet hairpin and adjacent α-helix. Our results suggest a new basis for the BlaR1 signaling mechanism, in which sustained L2-BlaRS contact mediates transmembrane signaling stimulated by changes in BlaRS dynamics upon binding of b-lactam antibiotics. We also found the b-lactam 2-(2'-carboxyphenyl)-benzoyl-6-aminopenicillanic acid (CBAP) is a useful tool to study the hidden conformational dynamics of BlaRS. Covalent acylation of the BlaRS active-site serine by CBAP shifts the active site motions to the slow exchange time scale. Slow conformational exchange allows the direct characterization of individual conformations in CBAP acylated BlaRS. Furthermore, the BlaRS active site has an architecture shared by many β-lactamases. This opens the possibility that CBAP can be a general probe to expose functional motions in β-lactam binding proteins with shared structural architecture.